OBJECTIVE

To study the possibility of the C17.2 neural stem cells (NSCs) differentiating into neural cells induced by serum-free condition medium of olfactory ensheathing cells (OECs) and to detect the cell viability of the differentiated cells.

METHODS

OECs were isloated and cultured from the olfactory bulbs of 3-day-old postnatal mouse to prepare serum-free condition medium of OECs. After C17.2 NSCs were cultured with H-DMEM/F12 medium containing 15% FBS and the cell fusion reached 80%, the 3rd passage cells were induced by serum-free condition medium of OECs in the experimental group, by H-DMEM/F12 in the control group, and non-induced C17.2 NSCs served as the blank control group. The growth condition of cells was observed with inverted microscope. After 5 days, the immunofluorescence staining [microtubule-associated protein 2 (MAP-2) and beta-tubulin-III] and Western blot (Nestin, beta-tubulin-III, and MAP-2) were carried out to identify the neural cells derived from NSCs. The cell viabilities were measured by MTT assay and the quantity of lactate dehydrogenase (LDH) release in the medium.

RESULTS

In the experimental group, the C17.2 NSCs bodies began to contract at 24 hours after induction, and the differentiated cells increased obviously with long synapse at 3 days after induction; in the control group, the cell morphology showed no obvious change at 24 hours, cell body shrinkage, condensation of nuclear chromatin, and lysis were observed at 3 days. The immunofluorescence staining showed that beta3-tubulin-III and MAP-2 of C17.2 NSCs were positive at 5 days after induction, and Western blot suggested that the expression of Nestin protein declined significantly and the expressions of beta-tubulin-III and MAP-2 protein were increased in the experimental group, showing significant differences when compared with those in the control group and blank control group (P < 0.05). The LDH release and the cell viability were 130.60% +/- 6.86% and 62.20% +/- 3.82% in the experimental group, and were 178.20% +/- 5.44% and 18.00% +/- 3.83% in the control group respectively, showing significant differences between 2 groups (P < 0.05). The LDH release and the cell viability of experimental group and control group were significantly lower than those of blank control group (100%) (P < 0.05).

CONCLUSION

Neurotrophic factors from OECs play an important role in inducing C17.2 NSCs differentiation into neural cells and keeping the viability of differentiated cells after induction.